α- and β-phosphorylated amines and pyrrolidines, a new class of low toxic highly sensitive 31P NMR pH indicators -: Modeling of pKα and chemical shift values as a function of substituents

Fourteen linear and cyclic alpha- and beta-aminophosphonates in which the P-atom is substituted by alkoxy groups have been synthesized and evaluated as P-31 NMR pH markers in Krebs-Henseleit buffer. pK(a) values varied with substitution in the range 1.3-9.1, giving potentially access to a wide range of pH, Temperature had a weak influence on pH and a dramatic increase in ionic strength slightly modified the pK(a) of the pyrrolidine diethyl(2-methylpyrrolidin-2-yl)phosphonate (DEPMPH). All compounds displayed a 4-fold better NMR sensitivity than inorganic phosphate or other commonly used phosphonates, as assessed by differences delta(b)-delta(a), between the chemical shifts of the protonated and the unprotonated forms. In isolated perfused rat hearts, a non-toxic concentration window of 1.5-15 mM was determined for three representative compounds, Using empirical linear relationships, the experimental values of pK(a), delta(a), and delta(b) have been correlated with the two-dimensional structure, i.e. the chemical nature of substituents bonded to the secondary amine and P-atom, The data suggest that DEPMPH and its cyclic and linear variants are ideal versatile P-31 NMR probes for the study of tenuous pH changes in biological processes.